Dendrimers for use in targeted delivery

ABSTRACT

The present invention provides cationic dendrimers for delivering bioactive molecules, such as polynucleotide molecules, peptides and polypeptides and/or pharmaceutical agents, to a mammalian body. The dendrimers disclosed herein are suitable for targeting the delivery of the bioactive molecules to, for example, the liver, spleen, lung, kidney or heart.

The present invention relates to the targeted delivery of bioactivemolecules in a mammalian body. In particular, the present inventionrelates to the use of cationic dendrimers for delivering polynucleotidemolecules, peptides and polypeptides and/or pharmaceutical agents to amammalian body, in particular, human.

The possibility of using genes as medicines to correct genetic disordersor treat cancers is hampered by the inability to efficiently delivergenetic material to diseased sites¹. A variety of viral and non-viralsystems are being used experimentally each with distinct advantages anddisadvantages. Viral systems² have been studied extensively and includea wide variety of viral types such as, retroviruses, adenoviruses,adeno-associated viruses, herpes simplex virus and the HIV basedlentivirus. All have various inherent disadvantages³ such as safetyconcerns and scale-up difficulties. Non-viral systems such as cationicliposomes⁴⁻⁶, cationic polymers^(7,8), cationic polymericvesicles^(9,10) and dendrimers¹¹⁻¹⁴ have thus been studied as genedelivery agents in an effort to circumvent some of the safety andproduction problems associated with viruses. As well as applications inhuman health, suitable gene transfer systems are commercially attractiveas in vitro molecular biology and in vivo transfection reagents forlaboratory use.

Dendrimers are synthetic 3-dimensional macromolecules that are preparedin a step-wise fashion from simple branched monomer units, the natureand functionality of which can be easily controlled and varied.Dendrimers are synthesised from the repeated addition of building blocksto a multifunctional core (divergent approach to synthesis), or towardsa multifunctional core (convergent approach to synthesis) and eachaddition of a 3-dimensional shell of building blocks leads to theformation of a higher generation of the dendrimers⁵¹. Polypropyleniminedendrimers start from a diaminobutane core to which is added twice thenumber of amino groups by a double Michael addition of acrylonitrile tothe primary amines followed by the hydrogenation of the nitriles⁵². Thisresults in a doubling of the amino groups⁵².

Polypropylenimine dendrimers contain 100% protonable nitrogens¹⁷ and upto 64 terminal amino groups (generation 5, DAB 64)^(15,16). Protonablegroups are usually amine groups which are able to accept protons atneutral pH. The use of dendrimers as gene delivery agents has largelyfocused on the use of the polyamidoamine^(11-13,18-25) and phosphorouscontaining¹⁴ compounds with a mixture of amine/amide or N—P(O₂)S as theconjugating units respectively with no work being reported on the use ofthe lower generation polypropylenimine dendrimers for gene delivery.Polypropylenimine dendrimers have also been studied as pH sensitivecontrolled release systems for drug delivery^(26,27) and for theirencapsulation of guest molecules when chemically modified by peripheralamino acid groups²⁸. The cytotoxicity²⁹ and interaction ofpolypropylenimine dendrimers with DNA³⁰ as well as the transfectionefficacy of DAB 64 has also been studied³¹.

Kabanov and others report that polypropylenimine dendrimers interactwith DNA via the surface primary amines only with no involvement of theinternal amine groups³⁰ while Gebhart and Kabanov report very low genetransfer activity with the 5^(th) generation polypropyleniminedendrimers DAB 64 in the easy to transfect COS cell line³¹. Theseworkers also report that DAB 64 is far too toxic above a dendrimer, DNAweight ratio of 0.62:1 (nitrogen to phosphate ratio of 4:1).Additionally Malik and others conclude that the cationic dendrimers asopposed to the anionic dendrimers are too toxic for parenteral usewithout further derivatisation with biocompatible groups such aspolyethylene glycol units²⁹.

The present invention is based upon the observation that, contrary toearlier reports, cationic dendrimers, such as polypropyleniminedendrimers, display suitable properties, such as specific targeting andlow toxicity, for use in the targeted delivery of bioactive molecules,such as genetic material. In addition, derivatives of the cationicdendrimer also display suitable properties for the targeted delivery ofbioactive molecules.

The chemical modification (derivatisation) of polypropyleniminedendrimers has been extensive with reports on the conjugation of aminoacid^(28,32), carboxylate^(33,34), acetyl³⁵,2-(hydroxy)propyltrimethylammonium²⁷, dimethyldodecylammoniumm³⁶,3,4,5-(ologoethylenoxy) benzoyl³⁷, alkanoyl thioglucose³⁸,thiolactosyl³⁹ groups as well as the conjugation of hydrophobic3,4-bis(decyloxy)benzoyl⁴⁰, palmitoyl⁴¹, pentafluorphenyl11-[4-(4-hexyloxyphenylazo)phenyloxy]undecanoyl⁴¹, adamantinecarboxyl⁴¹, decanoyl⁴², dodecyl⁴², polyisobutylene⁴³, stearoyl⁴⁴,ω-(4′cyanobiphenyloxy)alkyl⁴⁵, and oligo(p-phenylene vinylene)⁴⁶ groupsto the primary amino groups on the surface of polypropyleniminedendrimers.

In addition to the targeted delivery of genetic material, selectivetargeting of other bioactive agents, such as peptides/polypeptides andpharmaceutical agents, is sought.

It is thus one of the objectives of the present invention to providecationic dendrimers capable of targeted delivery of bioactive moleculesto a particular location in a mammalian body.

Accordingly, the present invention provides a composition for thedelivery of bioactive molecules to a target location in the body of arecipient, wherein said composition comprises a cationic dendrimer,and/or derivatives thereof, admixed with said bioactive molecule.

The term “cationic dendrimer” refers to a dendrimer molecule whichpossesses a positive charge at physiological pH. However, the dendrimerderivatives of the present invention may not in themselves be cationicas a result of the derivatisation.

The cationic dendrimers, or derivatives thereof, of the composition ofthe present invention may be derived from a core molecule comprising 2to 10 carbon atoms, such as 3 or 4 carbon atoms, and in particular 4carbon atoms with one or more functional groups which may, for example,be amine groups. It will be appreciated that, for example, the cationicdendrimers, or derivatives thereof, may be derived from a core moleculesuch as diaminoethane, diaminopropane or diaminobutane, and inparticular, diaminobutane.

The groups attached to the core molecule may, for example, includepropylamines Thus, the dendrimers may be polypropylenimine dendrimers,or derivatives thereof, and may possess a diaminobutane core.

The term “polypropylenimine dendrimer” is intended to refer todendrimers comprising a diaminobutane core with 1, 2, 3, 4 or 5generations of propylenimine molecules attached. The term encompassesDAB 4, DAB 8, 16, 32 and 64, DSAM 4, DSAM8, 16, 32 and 64, and QDAB4, 8,16, 32 and 64, HDAB4, 8, 16, 32, 64 and bolamphiphilic polypropyleniminedendrimers BDAB4, BDAB8, BDAB16, BDAB32, BDAB64. Although allgenerations of the compounds DSAM, QDAB, HDAB and BDAB comprise adiaminobutane core with propylenimine groups attached thereto, the term“DAB” as generally used herein is intended to refer to the underivatisedDAB8, 16, 32 and 64 compounds, unless otherwise indicated.

The term “generation” refers to the number of iterative reaction stepsthat are necessary to produce the compound. The number which follows thename or abbreviated name of the dendrimer, for example, 8, 16, 32 or 64refers to the number of surface groups on the dendrimer molecule itself,which amino groups may or may not be derivatised.

The cationic dendrimers of the composition of the present invention maybe modified by covalently binding derivatising groups, such ashydrophobic, hydrophilic or amphiphilic groups to the surface of thedendrimer or by attaching two dendrimer molecules to either end of ahydrocarbon chain with a carbon length of 8, 12, 14, 16 or 18 carbons togive bolamphiphilic dendrimers (said modified dendrimers referred toherein as “derivatives”). The number of derivatising groups may varyfrom one derivatising group per dendrimer molecule up to and includingderivatising all available surface or terminal groups on the dendrimermolecule, for example, derivatising all 16 surface groups of the DAB16molecule.

The amphiphilic derivative comprises a hydrophilic and a hydrophobicsegment. The hydrophilic segment may be derived from a phosphoglyceratemolecule, for example, glycerol 3-phosphate. The hydrophobic segment iscovalently bound to the hydrophilic segment, for example, via an esterlinkage. The hydrophobic segment is selected from any suitablehydrophobic group, for example, alkyl, alkenyl or alkynyl groups of 8-24carbons in length. Therefore, the hydrophobic segment plus ester linkagecan be defined as an acyl group. The amphiphilic derivative is attachedto the dendrimer by a linker molecule, such as polyethylene glycol (PEG)or a sugar unit such as muramic acid bound to the hydrophilic segment.The length of the PEG linker molecule may for example be in the range of1 to 120 ethylene glycol units, for example 50-100 and, for example,70-80, for example, 77. In particular the linker molecule may bepolyethylene glycol with a Mw of approximately 3,500. Alternatively, thelinker molecule may be an ester, amine or ether linkage for ordinaryhydrophobic modifications or a sugar molecule such as muramic acid. Inparticular, the derivative may be a phosphoglyceride such as aphosphatidyl ethanolamine, for example,distearoylphosphatidylethanolamine. The number of amphiphilicderivatives per dendrimer molecule may range from 1 derivatising groupper dendrimer molecule to derivatising all of the groups of thedendrimer (the generation of the dendrimer will determine the totalnumber of surface groups available for derivatising), and may be, inparticular, one group per dendrimer. Thus, the dendrimers of the presentinvention include generations 1, 2, 3, 4 and 5 of theamphiphilic-derivatised diaminobutane dendrimer referred to herein as1,2-diacyl-SN-glycero-3-phosphoethanolamine-N-[(polyethyleneglycol)-N-diaminobutanepolypropyleniminedendrimer-(NH₂)_(x)], where x=4, 8, 16, 32 or 64 (conveniently referredto as DSAM4, 8, 16, 32 or 64, respectively).

The hydrophobic derivative may be an alkyl, acyl, alkenyl, alkynyl oraryl group of 8-24 carbons in length. It is to be understood that theterm “hydrophobic” can encompass acyl groups when the chain length ofsuch acyl groups is 8 carbons or more and may, for example, be ahexadecanoyl group. The number of hydrophobic groups per dendrimermolecule may range from 1 derivatising group per dendrimer molecule toderivatising all of the groups of the dendrimer (the generation of thedendrimer will determine the total number of surface groups availablefor derivatising), and may be, in particular, one group per dendrimer.Thus, the dendrimers of the present invention include generations 1, 2,3, 4 and 5 of the hydrophobic-derivatised diaminobutane dendrimerreferred to herein as HDAB4, HDAB8, HDAB16, HDAB32, HDAB64.

The bolamphiphiles may consist of two molecules of any of the dendrimersDAB4, DAB8, DAB 16, DAB 32, DAB 64 linked to either end of an alkyl,acyl, alkenyl, alkynyl hydrophobic unit of 8 to 24 carbon chains inlength or alternatively linked by an aryl group and may be a C12bolamphiphile of DAB 4 or DAB 8. The term “bolamphiphiles” is understoodto refer to an amphiphilic molecule wherein the hydrophilic groups areseparated by the hydrophobic groups. Thus, the dendrimers of the presentinvention include C8-C16alkyl bolamphiphiles of dendrimers ofgenerations 1, 2, 3, 4 and 5 herein referred to as B8DAB4, 8, 16, 32 or64; B10DAB4, 8, 16, 32 or 64; B12DAB4, 8, 16, 32 or 64, B14DAB4, 8, 16,32 or 64 and B16DAB4, 8, 16, 32 or 64. The amino derivative may, forexample, be a tertiary amine or quaternary ammonium derivative, and inparticular a quaternary derivative comprising C1-C4 alkyl groups, suchas 3 methyl groups, covalently bound to a nitrogen atom on the surfaceof the dendrimer. The number of ammonium derivatives per dendrimermolecule may range from 1 derivatising group per dendrimer molecule toderivatising all groups of the dendrimer (the generation of thedendrimer will determine the total number of surface groups availablefor derivatising), and may be, in particular, all groups available forderivatising. Thus, the dendrimers of the present invention includegenerations 1, 2, 3, 4 and 5 of the quaternary ammonium-derivatiseddiaminobutane dendrimer referred to herein as quaternary ammoniumdiaminobutanepolypropylenimine dendrimer-[NH₂(CH₃)₃]_(x), where x=4, 8,16, 32 or 64 (conveniently referred to as QDAB4, 8, 16, 32 or 64,respectively).

The dendrimers in the present invention may also be derivatised withhydrophilic groups such as sugars, mono and oligohydroxy C1-C6 alkyl,mono and oligohydroxy C2-C6 acyl, C1-C2 alkoxy alkyl optionally havingone or more hydroxy groups substituted on the alkoxy or alkylene groups,amino acids, peptides of 1-200 amino acids in length and oligo orpoly-(oxa C1-C3 alkylene) such as polyoxyethylene comprising 1-120ethylene oxide units.

Target locations for the delivery of bioactive molecules include theliver, spleen, lung, kidney and heart. In particular, two of thedendrimers of the present invention studied, DAB16 and DSAM16, havedisplayed organ-specific targeting to the liver and spleen,respectively.

Therefore, the present invention also provides a composition for thedelivery of a bioactive molecule to the liver of a recipient, whereinsaid composition comprises the polypropylenimine dendrimer DAB16 admixedwith a said bioactive molecule. Additionally, the present inventionprovides a composition for the delivery of bioactive molecules to thespleen of a recipient, wherein said composition comprises thepolypropylenimine dendrimer DSAM16 admixed with a said bioactivemolecule.

The recipient may be a mammal, such as a human.

The terms “bioactive molecules” and “biologically active molecules” areintended to encompass polynucleotides, peptides/polypeptides and/orpharmaceutical agents. The term “polynucleotides” generally refers toDNA unless otherwise indicated but may include RNA, cDNA,oligonucleotides, plasmids etc. The term may also be usedinterchangeably herein with the terms “polynucleotide”, “gene”, “geneticmaterial” and “genetic sequence”. Such genes intended for expression arecommon to the field of gene therapy and include, but are not limited to,sense DNA or RNA for expressing a product in the target organ, orantisense DNA or RNA for reducing or eliminating expression of a nativeor introduced gene in the target organ. The term “peptide” refers to achain of 4 to 600 amino acids long, such as 4 to 200 amino acids longand therefore encompasses polypeptides and proteins, and includesenzymes and polypeptide hormones. Furthermore, peptides modified by, forexample, glycosylation, are also included in the present invention, asis a protein comprising two or more polypeptide chains each of length of4 to 600 amino acids long cross-linked by, for example, disulphidebonds, for example, insulin and immunoglobulins. The term“pharmaceutical agent” is intended to include any natural or syntheticcompound administered to a recipient in order to induce a physiologicalor pharmacological effect. Examples of such agents are anti-tumourdrugs, antibiotics, hormones, anti-inflammatory agents, antiparasiticagents, DNA vaccines, etc

The cationic dendrimers are admixed with the bioactive agents inpreparing the compositions of the present invention for delivery. Theterm “admixed” generally refers to the bioactive agent being associatedwith but not covalently bound to the dendrimer. The term is however alsointended to encompass covalently binding the bioactive agent to thedendrimer via any suitable reactive group on the dendrimer and theagent.

Where the bioactive agent is a polynucleotide molecule, the molecule isusually associated with, that is, not covalently bound to, the dendrimerto allow the polynucleotide to be expressed. However, it may also bepossible that expression of a covalently bound polynucleotide moleculecan occur, and therefore, these covalently bound polynucleotidemolecules are intended to be encompassed by the present invention.

In a yet further aspect, the present invention provides a pharmaceuticalformulation comprising a composition of the present invention, and apharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are well known to those skilled inthe art and include, but are not limited to, 0.1 M and preferably 0.05Mphosphate buffer or 0.8% (w/v) saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solutionsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers such as thosebased on Ringer's dextrose, and the like. Preservatives and otheradditives may also be present, such as, for example, antimicrobials,antioxidants, chelating agents, inert gases and the like.

Conveniently, the composition or pharmaceutical formulation of thepresent invention may include an agent which assists in forming acolloidal suspension, for example, 5% dextrose solution. Other agentswhich may be included are viscosity enhancing polymers such as alginatesand polyethyleneglycol polymers, buffering agents and mixtures ofaqueous and non-aqueous solvents in emulsions.

The present invention also provides the use of the composition orpharmaceutical formulation of the present invention for the delivery ofbioactive molecules to a target location in the body of a recipient.

In a further aspect, the present invention provides a method ofdelivering a bioactive molecule to a target location in the body of arecipient, which method comprises preparing a composition comprising acationic dendrimer, or derivative thereof, admixed with a said bioactivemolecule, and subsequently administering the composition to saidrecipient.

Although the dendrimers of the present invention display suitableproperties for the delivery of bioactive molecules in vivo, they arealso useful for transfecting mammalian cells in vitro. Thus, the presentinvention also provides a composition of the present invention fortransfecting mammalian cells with a bioactive molecule in vitro. Themammalian cells may, for example, be human cells.

The present invention still further provides1,2-diacyl-SN-glycero-3-phosphoethanolamine-N-[(polyethyleneglycol)-N-diaminobutanepolypropyleniminedendrimer-(NH₂)_(x)], where x=4, 8, 16, 32 or 64 (DSAM), and quaternaryammonium diaminobutanepolypropylenimine dendrimer-[NH₂(CH₃)₃]_(x), wherex=4, 8, 16, 32 or 64 (QDAB). The DSAM or QDAB dendrimers may, forexample, be second, third, fourth or fifth generation dendrimersreferred to herein as DSAM4, 8, 16, 32 and 64, and QDAB4, 8, 16, 32 and64.

The present invention also provides a method of preparing a compositionas described above, said method comprising admixing a cationicdendrimer, and/or derivatives thereof, and a bioactive molecule.

These and other aspects of the present invention will now be describedby way of example only, in conjunction with the accompanying Figures, inwhich:

FIG. 1 illustrates DAB 16 generation 3 polypropylenimine dendrimer, DAB64 contains 2 more generations of propylamines attached to thismolecule;

FIG. 2 illustrates DSAM 16, an amphiphilic derivative of DAB 16(DSPE-PEG-NHS+DAB16);

FIG. 3 illustrates BnDAB4, a bolamphiphile derivative of DAB4 (n=8, 10or 12 to give B8DAB4, B10DAB4, B12DAB4 respectively);

FIG. 4 illustrates QDAB 16, a quaternary ammonium derivative of DAB 16(CH₃₁+DAB16);

FIG. 5 illustrates Luciferase gene expression in vivo;

FIG. 6 illustrates liver targeting of gene expression by thepolypropylenimine dendrimers; and

FIG. 7 illustrates tumour gene expression after the intravenousadministration of DNA, PEI-DNA=the Exgen 500 formulation.

EXAMPLE 1

Methods

Synthesis of Modified Dendrimers

DSAM

To DAB 16 (Sigma Aldrich, Dorset, UK—3.52 g, 1.32 mmoles) dissolved inabsolute ethanol (70 ml) was added triethylamine (27 ml, 6.6 mmoles) andto this solution was added dropwise DSPE-PEG-NHS(distearoylphosphatdylethanolamine polyethylene glycolN-hydroxysuccinimide—500 mg, 0.11 mmoles, Shearwater Polymers) dissolvedin chloroform (25 mL) over 60 minutes. The reaction was left stirringprotected from light for 72 h. At the end of this time the reactionmixture was evaporated to dryness by rotary evaporation under reducedpressure at 50° C. The residue was redissolved in absolute ethanol (40ml), filtered and the filtrate evaporated to dryness. This latterresidue was dissolved in water (80 ml) and dialysed against 5 l of waterover 24 h with 6 changes. The dialysate was freeze-dried and thestructure confirmed by ¹H and ¹³C NMR.

QDAB

DAB 16, 32 or 64 (500 mg) was dispersed in methyl-2-pyrolidone (50 ml)for 16 h at room temperature with stirring. To the DAB dispersion wasadded sodium hydroxide (120 mg), methyl iodide (3 g) and sodium iodide(150 mg). The reaction mixture was stirred under a stream of nitrogenfor 3 h at 36° C. The quaternary ammonium product was recovered byprecipitation in diethyl ether followed by filtration. The solid waswashed with copious amounts of absolute ethanol (1 l) followed bycopious amounts of diethyl ether (500 ml). The washed solid was thendissolved in water (150 ml) and passed over an ion exchange column (1×6cm packed with 30 ml Amberlite IRA-93 Cl⁻ and subsequently washed withHCl—90 ml, 1 M followed by distilled water—500 ml until the eluate givesa neutral pH). The eluate obtained was freeze-dried and the structureconfirmed by both ¹H and ¹³C NMR.

DNA Condensation

Plasmid (pCMVsport β-gal or pCMV luciferase, Life Technologies, UK) wasgrown in E. coli and plasmid purification carried out using a QIAGENEndo-toxin free Giga Plasmid Kit (QIAGEN, Hilden, Germany) according tothe manufacturer's instructions. Purity was confirmed by agarose gelelectrophoresis⁴⁷. The reduced fluorescence of ethidium bromide (EthBr)was used to probe for DNA condensation by the polymers. EthBrfluorescence increases significantly (factor 40 compared to unboundEthBr) on intercalation with double stranded DNA⁴⁸. The electrostaticinteraction between the anionic DNA and cationic groups of the carrieron formation of the DNA—vesicle complex reduces the number of EthBrbinding sites, a process termed condensation, ultimately reducing thefluorescence intensity of the EthBr solution.

Complexes of DAB 16 and DSAM with DNA were prepared at various polymer,DNA weight ratios and at various time points the fluorescence intensity(λ_(excitation)=526 nm, λ_(emission•)=592 nm) of the complexesdetermined in the presence of EthBr (40 μg ml⁻¹). The DNA concentrationin the cuvette was kept constant (100 μg ml⁻¹) and the polymer solutionsin PBS (phosphate buffered saline, pH=7) and a solution of DNA in PBSserved as controls. The reduced fluorescence (F_(t)/F₀), was determinedfor each of the samples, where F_(t)=the fluorescence of the DNA,polymer complexes and F₀=the fluorescence of DNA alone.

In Vitro Cytotoxicity Assay

A human epidermoid carcinoma cell line (A431, ATCC CRL-1555) wasmaintained in Dulbecco's minimum essential medium (DMEM) supplementedwith 10% foetal calf serum (FCS) and 2 mM glutamine (GibcoBRL, UK) at10% CO₂ and 37° C.

Polypropylenimine dendrimer/dendrimer derivative formulationcytotoxicity was assessed by the measurement of the IC50 in a standardMTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromidethiazolyl blue—indicator dye) assay. Briefly, 96 well microtitre plateswere seeded with 5000 cells per well and incubated for 24 hrs. Dilutionsof the dendrimer/dendrimer—DNA formulations (100 μl) in tissue culturemedium (Opti-Mem) were incubated with the cells for 4 h. The sampleswere then replaced with fresh DMEM daily and incubated for 72 h. Afterthis period the indicator dye (50 μl, 50 mg ml⁻¹) was added to each welland incubated with the cells for 4 h in the dark. The medium andindicator dye were then removed and the cells lysed withdimethylsulphoxide (200 μl). After addition of Sorensen's glycine buffer(25 μl) the absorption was measured at 570 nm. Values were expressed asa percentage of the control to which no vesicles were added.

In Vitro Transfection

DNA Polymer Formulations

DAB and QDAB dendrimer—DNA (pCMVSport P-galactosidase) formulations weremade by mixing DNA and dendrimers in a 5% dextrose solution and allowingto stand for no longer than 15 minutes before use. The resultingcolloidal dispersion was sized by photon correlation spectroscopy(Malvern Instruments, UK).

Cell Culture

A431 cells (human epidermoid carcinoma cell line, ATCC, CRL-1555),maintained in Dulbecco's Minimal Essential Medium (DMEM, LifeTechnologies, UK) supplemented with foetal calf serum and L-glutamine (2mM) were seeded at a density of 10⁴ cells ml⁻¹ and 200 μL of the cellsuspension placed in 96 well flat bottomed plates. Cells were incubatedfor 24 h at 37° C. in 10% CO₂. Polymer—DNA complexes containing 200 μgml⁻¹ DNA (100 μl) and serum free medium (100 μl, OPTIMEM, Lifetechnologies, UK) were incubated with the cells for 4 h at 37° C. in 10%CO₂. Naked DNA served as the negative control while a formulationcomprising N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethylsulphate (DOTAP), DNA (5:1) served as the positive control. Boththe negative and positive controls were dosed at a level of 20 μg DNAper well while the level of DNA dosed with the dendrimers varied asindicated. After this time the incubation medium was replaced with DMEMculture media containing penicillin (100 U ml⁻¹) and streptomycin (0.1mg ml⁻¹) and once again incubated at 37° C. in 10% CO₂ for 48 h. Thecells were then washed in phosphate buffered saline (200 μl) and lysedwith 1×Passive Lysis Buffer (80 μL, Promega, UK) for 30 min. The celllysates were subsequently analysed for β-galactosidase expression asdescribed below.

β-Galactosidase Expression

To the assay buffer [50 μl (sodium phosphate buffer—200 mM, pH=7.3,magnesium chloride—2 mM, mercaptoethanol—100 mM,o-nitrophenol-β-galactopyranoside—1.33 mg ml⁻¹)] was added an equalvolume of the cell lysate from above within 96 well flat-bottomedplates. Samples were incubated for 1 h and the visible absorbance readat 405 nm on an automatic plate reader.

In Vivo Transfection

DNA-Polymer Formulations

Exgen 500 (linear polyethylenimine 22 kD, Euromedex, France) wasformulated with the luciferase reporter gene (pCMV luciferase) asdescribed by the manufacturers. Both DAB 16 and DSAM were dissolved in5% w/v dextrose by probe sonication (5 minutes with the instrument setat 15% of its maximum output) and mixed in a 5:1 weight ratio with DNA15 minutes prior to intravenous injection.

Animal Experiments

Groups of Balb/C mice (n=3) were injected via the tail vein with eitherthe DAB 16, DSAM, Exgen 500 or naked DNA formulations each containing100 μg of DNA in an injection volume of 200-400 μl. Exgen 500 and nakedDNA served as the positive and negative controls respectively. Organswere harvested 24 h later and quickly frozen in liquid nitrogen andstored at −80° C. until analysis could be performed on them.

Analysis for Luciferase

To the liver samples were added 5 μl of tissue lysis buffer (25 mM TrisHCl—pH=8.0, 2 mM DTT, 2 mM EDTA—pH 8.0, 10% w/v glycerol, 1% w/v TritonX-100, 1 Complete® protein inhibitor cocktail tablet per 50 ml ofbuffer) per mg of liver tissue. To the other organs was added 4 μl oftissue lysis buffer per mg of tissue. Organs were homogenised on ice inthe lysis buffer to form a slurry and the slurry incubated on ice for 15mins. The resulting slurry was centrifuged at 13,000 rpm to pellettissue debris and the supernatant removed to a fresh tube. To the pelletwas added another 50 μl of lysis buffer. The pellet was resuspended byhomogenisation and centrifuged once more at 13,000 rpm and thesupernatant removed and added to the previous supernatant sample.Supernatant samples were diluted 1:10 with the tissue lysis buffer and80 μl of this solution sampled and used for the luciferase assay whichwas carried out according to the protocol provided by Promega, UK.Protein estimation was carried out using the Sigma (Sigma-Aldrich, UK)bicinchoninic acid system (BC-1).

Results and Discussion

DNA binds electrostatically with the nitrogen rich dendrimers DAB 16,DAB 32 and DAB 64 (Table 2) and presumably with DAB 8. These compoundsbegin to condense DNA at a nitrogen to phosphate ratio of 1.47, 1.49 and1.45 respectively (Table 1) and a surface nitrogen to phosphate ratio of0.78, 0.77 and 0.74 respectively. TABLE 1 % Condensation of DNA by DABDendrimers Dendrimer, Dendrimer DNA weight ratio % DNA condensationafter 30 min DAB 16 5.00 90.3 2.00 93.9 1.00 96.2 0.50 96.0 0.25 94.10.13 42.8 QDAB 16 5.00 96.2 2.00 95.3 1.00 95.7 0.50 94.6 0.25 33.6 0.1319.2 DAB 32 5.00 94.4 2.00 96.9 1.00 96.7 0.50 96.1 0.25 87.2 0.13 21.8QDAB 32 5.00 96.7 2.00 96.9 1.00 96.4 0.50 43.9 0.25 12.6 0.13 7.5 DAB64 5.00 93.4 2.00 95.4 1.00 93.1 0.50 96.2 0.25 87.9 0.13 26.3 QDAB 645.00 96.0 2.00 96.2 1.00 95.4 0.50 46.0 0.25 15.3 0.13 11.9 DSAM 16 585.1 10 85.0 15 85.9 20 85.7It appears as if DNA does not bind only to the surface nitrogens ofpolypropylenimine dendrimers as reported³⁰ but also to nitrogenspossibly in the second shell of the dendrimer also. Condensation of DNAwith the polyamidoamine dendrimers has also been found to occur byelectrostatic means¹⁹. We propose that the polypropylenimine dendrimershave advantages over the polyamidoamine polymers for gene deliveryapplications simply due to the increased content of protonable nitrogenson the polypropylenimine polymers. There are also advantages associatedwith the polymer shape over linear polymers as DNA appears to interactwith the surface primary amines only, leaving the internal tertiaryamines available for the neutralization of the acid pH⁵⁰ within theendosomal/lysosomal compartment. The release of polyamidoamine carriedgenes by the endosome has been attributed to the protonation of theinternal tertiary nitrogens by endosomal protons which then results in aswelling of the endosome and the release of the DNA to the cytoplasm¹².Also the hydrolytic degradation of polyamidoamine dendrimer amide bondsin water or ethanol^(12, 13) increases transfection efficacy up to 50fold which the authors attribute to the increased flexibility of thepolymer on heat degradation. This increased flexibility is said to becrucial to the swelling of the endosome¹². However we propose thatsimply increasing the level of tertiary amines in the polymer availablefor neutralization of the endosomal/lysosomal pH improves transfectionirrespective of the flexibility of the polymer.

In vitro transfection efficacy with the polypropylenimine dendrimersreveals that protein expression obtained by using 20 μg of DNA in theDOTAP formulation is obtained using just 5 μg of DNA in the DAB 16formulation and there is no significant difference between the use of 15μg DNA in the QDAB 16 formulation and 20 μg DNA in the DOTAP formulation(Table 2). TABLE 2 The in vitro transfection of DAB dendrimers in theA431 cell line β-galactosidase Formulation DNA dose per well (μg)expression* DAB 8, DNA (3:1 g g⁻¹) 20 101.3 15 102.6 10 80.1 5 45.1 DAB8, DNA (5:1 g g⁻¹) 20 108.7 15 96.5 10 73.1 5 59.2 DAB 8, DNA (10:1 gg⁻¹) 20 82.0 15 76.4 10 78.6 5 71.7 DAB 16, DNA (1:1 g g⁻¹) 20 54.2 1540.0 10 38.3 5 33.7 DAB 16, DNA (3:1 g g⁻¹) 20 23.9 15 40.7 10 57.7 556.5 DAB 16, DNA (5:1 g g⁻¹) 20 18.3 15 24.1 10 88.4 5 100.8 QDAB 16,DNA (1:1 g g⁻¹) 20 54.2 15 40.0 10 38.3 5 33.7 QDAB 16, DNA (3:1 g g⁻¹)20 76.4 15 70.4 10 45.3 5 13.8 QDAB 16, DNA (5:1 g g⁻¹) 20 42.3 15 50.810 48.5 5 45.5 DSAM 16, DNA (5:1 g g⁻¹) 20 28.6 15 35.2 10 25.1 5 26.7DSAM 16, DNA (10:1 g g⁻¹) 20 26.1 15 22.2 10 22.3 5 24.3 DSAM 16, DNA(15:1 g g⁻¹) 20 21.1 15 21.6 10 21.4 5 21.9 DAB 32, DNA (3:1 g g⁻¹) 2012.2 15 16.3 10 17.6 5 16.6 QDAB 32, DNA (3:1 g g⁻¹) 20 13.8 15 23.2 1033.1 5 21.3*% Expression relative to expression obtained for optimum DOTAP (DOTAP,β-galactosidase reporter DNA ratio = 5:1) formulation on dosing cellswith 20 μg DNA. % protein expression relative to DOTAP formulationobtained with 20 μg naked DNA alone = 25.15%

Transfection with DAB 8 is also slightly superior to that obtained withDOTAP (Table 2). This indicates a superior gene transfer activity forthe DAB 8 and DAB 16 dendrimers. DAB 4 is currently being tested in ourlaboratories. For the DAB 16 formulation, transfection appears to beoptimum when using DAB 16 complexes with DNA at a nitrogen to phosphateratio of 30:1, forming complexes of 150 nm in size. Transfection withDAB 8 is also optimum at a nitrogen to phosphate ratio of 30:1.Transfection with polyamidoamine dendrimers is optimum when low-densitysoluble material is formed at a nitrogen to phosphate ratio of 20:1¹⁹.

DAB 8 the most transfection efficient molecule studied to date in thepolypropylenimine dendrimer class is also the least toxic, exhibiting anIC50 almost 6× higher than DOTAP (Table 3). TABLE 3 In vitrocytotoxicity against the A431 cell line Formulation IC50 (μg ml⁻¹) DAB 8352.4 DAB 8, DNA (5:1 g g⁻¹) 669.4 DAB 16 38.9 DAB 16, DNA (5:1 g g⁻¹)36.0 QDAB 16 44.6 QDAB 16, DNA (3:1 g g⁻¹) 129 DAB 32 5.7 DAB 32, DNA(3:1 g g⁻¹) 5.8 QDAB 32 11.2 QDAB 32 (3:1 g g⁻¹) 33 DOTAP 62

The data in Table 3 indicate that the complex formed by DNA and thequaternised molecule (QDAB 32 and QDAB 16) is less toxic than thatformed by DNA and the unquaternised molecule. The quartenised moleculeQDAB 16 is as active as DOTAP at the 20 μg DNA dose and QDAB 32 showsslight activity as a gene transfer agent at the 10 μg dose level (Table2) while DAB 32 is inactive. It is envisaged that QDAB 8 will produce agene transfer formulation with good biocompatibility and also with noloss of activity when compared to the unquaternised parent polymer.

DAB 16 and DSAM are efficient deliverers of DNA to tissues in vivocomparing favourably to the commercial product Exgen 500 and with theadded ability of being able to target the liver (DAB 16) and spleen(DSAM 16) more effectively than Exgen 500 (Table 4, FIG. 5). TABLE 4 Invivo luciferase expression obtained in the mouse model % Luciferaseexpression relative to Exgen 500* Formulation Lung Liver Kidney HeartSpleen DAB 16 48.9 762.6 28.6 73.2 100 DSAM 16 25.8 264.3 11.3 58.2599.2 DNA Alone 9.2 43.6 10.6 33.0 97.0*% luciferase expression relative to that obtained with linear PEI (Mw =22 kD, Exgen 500 ®) on intravenous injection of 50 μg DNA DAB 16 (DAB16, DNA weight ratio = 5:1) and DSAM 16 (DSAM 16, DNA weight ratio =5:1) formulations. Exgen 500 formulation consists of linear PEI, DNAweight ratio = 6:1.

CONCLUSIONS

In summary the lower generation polypropylenimine dendrimers (DAB 8 andDAB 16) show improved biocompatibility when compared to DOTAP andtransfection activity which is at some dose levels superior to thatobtained with DOTAP. Additionally DAB 16 may be used to target theliver, and DSAM 16 used to target the spleen in vivo.

EXAMPLE 2

Materials

All polypropylenimine dendrimers, glucose, were obtained fromSigma-Aldrich, UK. Phenyl methyl sulphonyl fluoride (PMSF), proteaseInhibitor cocktail and phosphate buffered saline tablets, isopropanoland maltose were all supplied by Sigma Aldrich, UK.9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)-D-galactopyranoside(DDAO) was purchased from Molecular Probes. Exgen 500 (linearpolyethylenimine, Mw=22 kD) was obtained from Euromedex, France. Passivelysis buffer was supplied by Promega, UK. pCMV-beta gal DNA was obtainedfrom Life Sciences/Invitrogen and propagated in E. Coli as previouslydescribed⁹.

Methods

Groups of healthy female Balb-C mice (n=3) were injected intravenouslywith either DAB8-DNA, quaternary ammonium DAB8 (Q8)-DNA, DAB16-DNA orDAB32-DNA or Exgen 500 (linear polyethylenimine)-DNA. Formulations ofthe Dendrimer or Exgen 500, DNA Complexes (200 μl) dispersed in glucose5% w/v containing 100 μg DNA were injected into each mouse and thedendrimer, DNA weight ratios were as follows: DAB8, Q8 and DAB 16 wereall administered at a dendrimer, DNA weight ratio of 5:1. DAB32 wasadministered at a dendrimer, DNA weight ratio of 3:1. Exgen 500 wasadministered in accordance with the manufacturers instructions.

Mice were killed after 24 h and their lungs and livers removed andfrozen in liquid nitrogen until an assay for β-galactosidase could beperformed. For the assay, 1 g of organ was made up to 2 mL with aprotease lysis buffer. The protease lysis buffer consisted of a)Protease lysis buffer 5× (1 mL), b) Phenyl methyl sulphonyl fluoride(PMSF) (50 mM in methanol, 200 μL), c) Protease Inhibitor cocktail (100μl) and water (3.7 mL).

1×10⁶ A431 cells dispersed in 0.1 ml phosphate buffered saline (pH=7.4,PBS) were implanted subcutaneously in each flank of CD-1 female nudemice. 4 days after the injection, the tumours were palpable (around 2mm). 8 days after the injection, the size of the tumours increased(around 5 mm) and the blood vessels were more visible. The animals weredosed 11 days after tumour implantation. Mice (n=4) were injectedintravenously with 50 □g DNA as naked DNA, DAB16-DNA, Exgen500-DNA alldispersed in 200 μL 5% w/v dextrose. Control animals were injected with5% w/v dextrose. Animals were killed 24 h later and tumours were excisedand immediately frozen in liquid nitrogen. For the assay each tumour wasadded to 0.3 mL of the Protease lysis buffer. Organs contained in thebuffer were homogenised and 100 μl of the homogenised organ dispersionwas added to 300 μl of the assay reagent. The assay reagent consistedof: a) DDAO 5 mg mL⁻¹ in DMSO (15 μL), b) PMSF 50 mM in methanol (20μL), c) maltose 20% w/v in PBS (100 μl), d) Protease inhibitor cocktail(15 μL), e) PBS (150 μl). The samples were incubated for the appropriatetime (45-90 min.) at 37° C. 200 μl of this mixture was warmed at 95° C.for 2 min, in order to stop the β-galactosidase reaction and todenaturate the proteins which could interfere with the assay. 800 μlisopropanol was then added to the dispersion. The mixture obtained wasvortexed to homogeneity and shaken for 20 min in the dark. Thedispersion was then centrifuged for 4 min, at 13000 rpm. 500 μl of thesupernatant was then added to 500 μl distilled water and thefluorescence read on a Beckman LS-50B fluorimeter (λ_(Exc): 630 nm,λ_(Em): 658 nm, slit: 2.5 nm). The amount of enzyme was then quantifiedusing a β-galactosidase standard.

Results and Discussion

DAB16, Q8 and DAB 32 all resulted in liver targeting when compared tothe commercial formulation Exgen500 (FIG. 6). DAB 16 resulted in moregene expression in the tumours when compared to Exgen500 (FIG. 7).

This data provides further support that polypropylenimine dendrimerstarget the liver and produce higher gene expression in tumour tissuewhen compared to commercial formulations where, for example, non-viralgene delivery systems target the lung^(8,53). Targeting the liver islikely to prove useful in the treatment of liver enzyme deficiencies andliver tumours. FIG. 7 illustrates that high expression in tumours may beobtained with the polypropylenimine dendrimers.

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1-33. (canceled).
 34. A composition for the delivery of a bioactivemolecule to a target location in the body of a recipient, saidcomposition comprising a cationic polypropylenimine dendrimer comprisinga diaminobutane core with 1, 2, or 3 generations of propyleniminemolecules attached, admixed with the bioactive molecule.
 35. Thecomposition according to claim 34, wherein the cationic dendrimers aremodified by covalently binding derivatising groups.
 36. The compositionaccording to claim 35, wherein the cationic dendrimers are derivatisedusing groups selected from the group consisting of hydrophobic,hydrophilic and amphiphilic groups.
 37. The composition according toclaim 35, wherein the cationic dendrimers are derivatised by binding twodendrimer molecules to either end of a hydrocarbon chain.
 38. Thecomposition according to claim 37, wherein the length of the hydrocarbonchain is selected from the group consisting of 8, 12, 14, 16 and 18carbons.
 39. The composition according to claim 37, wherein thederivatised cationic dendrimer is a bolamphiphilic dendrimer.
 40. Thecomposition according to claim 39, wherein the number of derivatisinggroups is from one derivatising group per dendrimer molecule up to andincluding derivatising all available surface or terminal groups on thedendrimer molecule.
 41. The composition according to claim 36, whereinthe amphiphilic derivative comprises a hydrophilic and a hydrophobicsegment.
 42. The composition according to claim 41, wherein thehydrophilic segment is derived from a phosphoglycerate molecule.
 43. Thecomposition according to claim 41, wherein the hydrophobic segment iscovalently bound to the hydrophilic segment via an ester linkage. 44.The composition according to claim 41, wherein the hydrophobic segmentis selected from the group consisting of alkyl, alkenyl and alkynylgroups of from 8 to 24 carbons in length.
 45. The composition accordingto claim 41, wherein the amphiphilic derivative is attached to thedendrimer by a linker molecule selected from the group consisting ofpolyethylene glycol (PEG) and a sugar molecule.
 46. The compositionaccording to claim 45, wherein the length of the PEG linker molecule isfrom 1 to 120 ethylene glycol units.
 47. The composition according toclaim 45, wherein the linker molecule is a phosphoglyceride.
 48. Thecomposition according to claim 41, wherein the number of amphiphilicderivatives per dendrimer molecule is from 1 derivatising group perdendrimer molecule to derivatising all of the groups of the dendrimer.49. The composition according to claim 34, wherein the bioactivemolecule is selected from the group consisting of polynucleotides,peptides, polypeptides and pharmaceutically active agents.
 50. In amethod of transfecting mammalian cells in vitro with a composition, theimprovement comprising transfecting said cells in vitro with acomposition according to claim
 34. 51. A pharmaceutical formulationcomprising the composition of claim 34 and a pharmaceutically acceptablecarrier.
 52. A method of delivering a bioactive molecule to a targetlocation in the body of a recipient, comprising administering thecomposition of claim 34 to said recipient.
 53. The method according toclaim 52, wherein the target location is selected from the groupconsisting of the liver, spleen, lung, kidney and heart.
 54. The methodaccording to claim 52, wherein the composition is for the delivery of abioactive molecule to the liver of the recipient, and wherein saidcomposition comprises the polypropylenimine dendrimer DAB16 admixed withsaid bioactive molecule.
 55. The method according to claim 52, whereinthe composition is for the delivery of a bioactive molecule to thespleen of the recipient, and wherein said composition comprises thepolypropylenimine dendrimer DSAM16 admixed with said bioactive molecule.56. The method according to claim 52, wherein the recipient is a human.57. A method of preparing the composition of claim 34, comprisingadmixing a polypropropylenimine dendrimer comprising a diammobutane corewith 1, 2 or 3 generations of propylenimine molecules attached, and/orderivatives thereof, and at least one bioactive molecule.